This invention relates to arrays, and more particularly to capture arrays using polypeptide, peptide and protein fragments for the detection of other proteins or amino acid oligomers.
Various arrays have been designed for use in genetic testing, screening and diagnostics. Most of the arrays that have been developed include the use of defined regions having biopolymers or nucleotides arranged in a predetermined configuration on a defined substrate. Most importantly, these arrays when exposed to a particular analyte will exhibit a pattern indicative of the presence and concentration of a particular component, peptide or oligonucleotide. Array binding patterns using polynucleotides and/or peptides can be detected by using a variety of suitable fluorescent target labels. Once bound to the array, the overall fluorescence pattern on the array is determined and the target labels can then be quantified and observed.
Arrays using proteins, peptides and protein fragments have been gaining much attention. Using these types of arrays, various techniques can be used to identify and capture various proteins of interest. Construction of this type of array can be used to effectively capture proteins of interest. Arrays for capturing proteins can be made using polypeptide capture agents such as antibodies, antibody fragments, or polypeptides selected from randomized or combinatorial libraries by means of phage display, ribosome display, or mRNA-protein fusion. However, there is a particular difficulty and disadvantage in using an array of amino acid oligomers to detect other proteins. In the first instance, there is the possibility of cross reactivity between the capture probe and the wrong protein. However, more importantly, the array needs to distinguish the protein-capture agent complex from the capture agents themselves.
A number of techniques have been developed for actually distinguishing the capture agents from the target agents or proteins, or from the capture agent/target complex. For instance, measurements can be done by using techniques such as ellipsometry, surface plasmon resonance, or microbalances based on surface acoustic waves. Other approaches require tagging or labeling the target agent or protein with a reagent that can be detected. For instance, detection of tags or labels can be done using optical or electrochemical technology. These latter tagging methods, which include direct optical techniques such as fluorescence, as well as indirect visualization by means of a biotin tag and subsequent avidin complexation, require that the target proteins be labeled with a tag before the assay is performed. Labeling the bound protein after the assay is performed is not feasible because the capture agents, that are amino acid oligomers, (e.g. antibodies) will also be labeled. Thus, the array feature with or without a bound target present will most likely then give a false positive response or reading.
A number of solutions have been proposed to the labeling problems discussed above. For instance, labeling the target molecules before the assay is performed gets around the problem of false positive responses that is due to the undesired reaction of the tag with the capture agent. However, this has many disadvantages. For example the unreacted tag can interfere with the binding assay, extra purification may need to be performed, or the modification of the protein with a tag may seriously affect the binding behavior of the protein (i.e. the Kd or selectivity may be altered or affected). It would be desirable therefore, to provide a means for detecting a target or target protein using polypeptide probes, particularly in the form of an addressable array, which can provide good binding affinity and specificity for a target protein or polypeptide. It would also be desirable to combine the strengths of the above technology to construct an array based system or methodology for protein analysis that is rapid, efficient and that is amenable to protein monitoring applications. In addition, it would be desirable to develop capture agents for use in an array that can detect amino acids, peptides or proteins (i.e. oligomer targets) without the need for pre-derivatization of the target. It would also be preferable to have a binding assay that can be done using unmodified targets, where detection is done after the targets have bound and after unbound targets have been washed away. Lastly, it would be desirable to have a system where specific tags can be used to react with the side chains on the target proteins, while the features of the array, consisting of amino acid oligomers, remain unchanged.
The references cited in this application, are incorporated in this application by reference. However, cited references are not admitted to be prior art to this application.
The invention is a method for detecting the presence of a target protein molecule using a polypeptide target probe. The target protein is not derivatized or pre-modified. Binding takes place between the target protein and the capture agent. The target protein is then modified for detection, while the capture agent remains unmodified for detection. The apparatus of the present invention includes an addressable array comprising a number of polypeptide capture agents designed to bind a particular target. The capture agents do not contain one or more defined amino acids with a modifiable side chain that may be used for detection. The target protein contains one or more of these same amino acids with modifiable side chains used in detection.